KR100329259B1 - manufacturing process of ginseng saponins - Google Patents

Abstract

The present invention is to prepare a rare anti-cancer saponin by hydrolysis of ginseng saponin ginseng using a saponin glucoside degrading enzyme.

Common cellulose enzyme (cellulase) and hemi-cellulose enzyme (Hemi-cellulase) can hydrolyze the polysaccharide pull (多糖 體 糖 基), but it is difficult to break the saponin pull (糖 基). The reason is that saponins have non-glycol aglycone groups, so only special saponin degrading enzymes can decompose saponins. In the present invention, saponin degrading enzyme, for example, saponin alpha-glucosidase (alpha-glucosidase) to decompose the saponin suffix of the protopanaxadiol-type ginsenosides (high base) Ginsenoside Rh2 can be obtained with a yield of 40-80%.

In addition, ginsenoside Rg1 may be prepared by decomposing ginsenoside Rg2 succinic acid with alpha-rhamnosidase (also, ginsenoside Rg1 may be prepared by hydrolyzing ginsenoside Re). Yield is 40-80%. In addition, the mixed ginseng saponin is reacted with a saponin degrading enzyme, thereby preparing a mixed saponin having a high content of anti-cancer saponins ginsenosides Rh2 and Rh1. In addition, the ginseng that produces saponin-degrading enzymes can be cultured in ginseng to make ginseng powder, ginseng fragments and other products with high content of anti-cancer saponins ginsenosides Rh2 and Rh1.

Description

A method for producing rare ginseng saponins by changing ginseng saponin extract with an enzyme {manufacturing process of ginseng saponins}

The present invention relates to a method for producing rare ginseng saponin having high physiological activity by changing ginseng saponin extract with an enzyme.

Ginseng is a plant taxonomy belonging to the genus Ginseng of the family Araliaceae, with four species and several other varieties. For example, Panax ginseng, Panax quinquefolium, Panax notoginseng, Panax notoginseng, Bamboo shoot, P. japonicus and others There is. Ginseng saponins are produced according to the structure of saponin aglycone, Protopanaxadiol-type gisenosides, Protopanaxatriol-type ginsenosi-des, and oleolinic acid ( I) It can be classified into three types of oleanolic acid-type ginsenosides. Ginsenoside derivatives, the pharmacologically active saponins unique to ginseng, are saccharides such as glucose, rhamnose, arabinose or xylose in the trimarpenic triterpenoids, Protopanaxadiol and Protopanaxatriol. Is a unique saponin that exists only in plants of the genus Panax, and the saponin content of ginseng is about 4-6% (6-year ginseng). To date, 34 kinds of derivatives from Korean ginseng have been identified, and it is already known that ginseng saponins have different pharmacological effects depending on the type of sugars bound to aglycone or the number or location of the sugars. In particular, many studies have been published on the pharmacological effects of major saponins contained in fresh ginseng and white ginseng, and recently, trace saponins present only in red ginseng. In particular, ginsenosides Rh1 and Rh2 which can be prepared according to the present invention have Red ginseng saponin with anticancer effect is the most potent inducing tumor cell regeneration in F9 teratocarcinoma cultures (Lee et al., Proc. 6th Intl. Ginseng symp., 127, 1993). Side Rh2 is Morris liver cancer cells (Odashima et al., Europ. J. Cancer, 15, 885, 1979) and B16 melanoma (black tumor cells), MK-1 (gastric cancer cells) (Matsunaga et al., Chem. Pharm. Bull., 38 , 3480, 1990) and ovarian cancer cells (HRA) (Kikuchi et al., Anticancer Drugs. England, 2, 63, 1991) have been reported to exhibit potent proliferative inhibitory activity on cultured cancer cells. (Matsunaga et al., Chem. Pharm. Bull., 37, 1279, 1992) In addition, in vivo studies of transplanting ovarian cancer cells (HRA) have shown toxicity and side effects when combined with anticancer drug cisplatin or when administered alone. Little is known (Tode et al., J. Cancer Res, Clin. Oncol., 120, 24, 1993). The effects of suppressing ovarian cancer and prolonging survival were similar to cisplatin. (Kikuchi et al., Anticancer Drugs, England, 2, 63, 1991) On the other hand, red ginseng is prepared by steam treatment of ginseng, and in this process, the glycoside binding to the C-20 position of the chemically structurally unstable ginsenoside aglycone is lost. Easily hydrolyzed. Therefore, red ginseng contains prosapogenin (ginsenosides Rh1, Rh2, Rg2, Rg3, etc.) much higher than ginseng or white ginseng. In particular, ginsenoside Rh2, an anti-cancer saponin, does not exist in white ginseng (Kitagawa et al., 103, 612, 1983). As such, the microsaponins Rh1 and Rh2 are chemically degraded (De Mayo et al., Canad. J. Chem., 43, 2033, 1965) or enzymatically (Kitagawa et al., Tetrahedron Letters, 30, 2283, 1974), In addition, there have been many attempts to manufacture by the method using the synthesis of glycosides (Korea Patent Publication No. 1995-0007250, etc.), but it has to go through several steps in the manufacturing process, due to the loss of the target component or use of a catalyst unsuitable for food It was difficult to industrialize and, above all, mass production was impossible due to low yields, for example, in general, cellulase and hemi-cellulase ( For example, fibrin alpha-glucosidase, fibrin alpha-lamnosidase) is difficult to hydrolyze ginseng saponin extract. The reason is that, unlike fibrin, saponin aglycone has non-glycolic aglycone, so only a specific saponin degrading enzyme can hydrolyze saponin hap to produce rare saponin. Therefore, the present invention overcomes these problems and partially extracts ginsenosides Ra, Rb, Rc, Rd and Rg3 which are high in ginseng by enzymatic methods. By decomposing, high value-added ginsenoside Rh2 having high anticancer properties can be prepared. The same method is used to hydrolyze saccharides such as Re, Rf, Rg1 and Rg2, which are the protofaxaxatriol-based ginsenosides. Ginsenoside Rh1 can be prepared.

The present invention is to produce a rare saponin by hydrolyzing the saponin of saponin high in ginseng with saponin glucoside degrading enzyme.

All kinds of ginseng belonging to the genus Ginseng, such as panax ginseng, using saponin degrading enzymes such as alpha-glucosidase and alpha-lamnosidase, which can hydrolyze saponin extracts. (Panax ginseng), Panax notoginseng, Panax quinquefolium, Panax japonicus, Panax pseudoginseng, or the leaves or tissue cultures of their plants. Or, using processed red ginseng, hydrolysis of saponin molecules partially produces new rare saponins.

Saponin-degrading enzymes can be obtained by fermentation from microorganisms such as bacterium, mold, yeast, etc., and also ginseng and malt. , Can be extracted from the bran (bran).

Ginsenoside Rh2, a rare saponin, can be prepared by hydrolyzing the protopanaxadiol-based ginsenosides with saponin alpha-glucosidase.

Ginsenoside Rg1 can be prepared by hydrolyzing ginsenoside Re, a protoparanaxatriol ginsenoside, with saponin alpha-rhamnosidase, and ginsenoside Rh1 by hydrolyzing ginsenoside Rg2.

By treating ginseng mixed saponins with saponin degrading enzyme, mixed saponins with high content of rare saponins ginsenosides Rh2 and Rh1 can be obtained. In addition, the bacteria that produce saponin-degrading enzymes are directly cultured in ginseng powder and ginseng slices to obtain ginseng powder, ginseng slices, and other products having a high content of rare saponins. The explanation is as follows.

-Example 1-

The culture medium containing 3% corn flour, 1% ginseng extract and 0.01% MgSO4 was incubated for 30-40 hours at 60oC under aerobic bacteria (High Bacillus sp.). By removing the cells with a separator to precipitate the enzyme protein (酵素 蛋白) with 50-80% ethyl alcohol (ethyl alcohol) solution, the protein collected by lyophilization can be prepared saponin degrading enzyme.

1 g of the saponin degrading enzyme is mixed with 140 ml physiological saline, and 3 g of ginsenoside Rg3 is dissolved in 20 ml acetic acid (醋酸; 0.02 M, pH 5.0) and 40 ml ethyl alcohol. After mixing the two solutions, the reaction is allowed to react for 12 hours while stirring at 60 ° C. at low speed. After the reaction, 800 ml of ethyl alcohol was added and filtered to obtain a protein precipitate. Wash the precipitate with excess ethyl alcohol. The filtrate is evaporated to dryness under reduced pressure. This gives 1.5-2 g of saponin with a ginsenoside Rh2 content of 50-85%.

Example 2

Ginseng is crushed to 15 necks (3) and then 3 times the volume of sodium acetate (0.02M, pH5-6) solution is added. Extraction for 1-3 hours with stirring at 40 ° C. at low speed. After that, the impurities are removed by filtration, ethyl alcohol is added to the supernatant, and the enzyme protein is precipitated. The protein is collected and lyophilized to obtain saponin degrading enzyme. Ginsenoside Rh2 can be obtained by treating ginsenoside Rg3 with the saponin degrading enzyme thus prepared by the method of Example 1.

Example 3

Add bacteria to the culture medium containing 3% wheat bran and 1% ginseng powder, and incubate at 28-30oC for 60-80 hours to remove the cells. Precipitate enzyme protein with -80% ethyl alcohol solution, collect proteins, dissolve in 1/10 volume of sodium acetate solution (0.02M, pH5.0) and remove impurities by centrifugation. Can be obtained.

3 g of ginsenoside Rd is dissolved in 100 ml of acetic acid solution (0.02 M, pH 5.0) and 50 ml of the enzyme solution is added thereto. Reaction at 40 ° C. for 6-24 hours results in ginsenoside F2. Then, butanol (Buthanol) of 1/3 volume is added and the saponin is extracted three times and evaporated to dryness under reduced pressure (減壓). Organic acids (such as nitric acid, acetic acid, hydrochloric acid, sulfuric acid, etc.) hydrolyze the carboxylic acid at the 20th carbon position of the ginsenoside F 2 molecule to convert it into ginsenoside Rh 2. When this is separated by a silica gel column separation method, 0.5-1.4 g of ginsenoside Rh2 may be prepared. In the same manner, ginsenosides Rh2 can be prepared from the protoparnaxadiol-based ginsenosides Rb1 and Rc.

Example 4

After removing the mycelium from the Korean culture, put the ethyl alcohol to 50-80%. Precipitate the enzyme protein (酵素 蛋白), collect the protein and dissolve in sodium acetate solution (0.02M, pH5.0). Alpha-rhamnosidase was isolated and lyophilized using a DEAE-Cellulose column.

After dissolving 10 g of the protoparnaxadiol-based ginsenoside Re in 200 ml sodium acetate solution (0.02 M, pH 5.0), the mixture was added with alpha-rhamnosidase and reacted at 40 ° C. for 12 hours. Then, the mixture was heated at 100 ° C. for 30 minutes, filtered to separate protein precipitates, adsorbed onto Macroporous Resin AB-8, washed with 500 ml of 70% ethyl alcohol, and then dried under reduced pressure. Evaporation to dryness yields 4-5.5 g of ginsenosides Rg1. By treating 10 g of ginsenoside Rg2 in the same manner, 4-6 g of ginsenoside Rh1 can be obtained.

Example 5

Bacillus sp. After dissolving 0.3 g of enzyme and 10 g of mixed saponin made by fermenting JF bacteria in 200 ml of sodium acetate solution (0.02 M, pH 5.0), the mixture was reacted at 40 ° C. for 12 hours and quantified by high performance liquid chromatography (HPLC). As a result, more than 80% of the saponins lost at least one strain, increasing the content of rare anticancer saponins ginsenosides Rh1 and Rh2 by 50-200 times. The reaction solution was heated to remove enzyme protein, adsorbed onto 200g air resin AB-8, washed with 500ml of 90% ethyl alcohol, and then dried under reduced pressure. Mixed saponins with high side Rh1 and Rh2 contents can be obtained.

Example 6

Inoculate Korean ginseng into 50 g of ginseng slices or powders with 50-150% water content at 106 -108 C.FU / ml, and incubate 2-3 days at 28-38 o C. You can get this high ginseng. As a result of quantitative analysis by high performance liquid chromatography (HPLC), 40-80% of saponins lost at least one core, which contained the rare anticancer saponins, ginsenosides Rh1 40- 60 mg, Rh2 50-80 mg. Ginseng pieces or powder can be obtained.

The saponins or mixed saponins prepared by the present invention can be used as raw materials for foods, cosmetics, medicines, reagents and industrial products.

Claims (6)

A saponin degrading enzyme containing alpha-glucosidase and alpha-lamnosidase hydrolyzes ginseng saponin of the ginseng genus, and the saponin molecules are partially hydrolyzed by hydrolysis. Method for producing ginseng saponin, characterized by converting saponins present in trace amounts or not present in red ginseng into saponins present in red ginseng and wild ginseng According to claim 1, saponin degrading enzyme can be obtained by fermentation method in bacteria (bacterium), mold, yeast, and also ginseng, malt, wheat vein Method using an enzyme that can be extracted in (bran). The method according to claim 1, wherein ginsenoside Rh2 can be prepared by hydrolyzing the protopanaxadiol-based ginsenoside with saponin alpha-glucosidase. The method according to claim 1, wherein ginsenoside Rg1 is produced by hydrolyzing the protopanaxadiol-based ginsenoside Re with saponin alpha-rhamnosidase, and hydrolyzing the ginsenoside Rg2 to produce ginsenoside Rh1. The method of claim 1, wherein ginsenosides Rh2, Rh1 content of the mixed saponins are prepared by treating ginseng mixed saponins with saponin degrading enzyme. According to claim 1, bacteria that produce saponin-degrading enzymes are directly cultured in ginseng powder and ginseng slices, and ginseng powder, ginseng concentrate, ginseng fragment, which are not present in fresh ginseng and white ginseng, or have a small amount of saponin. How can I get ginseng products?